Essential role of gastric gland mucin in preventing gastric cancer in mice

信州大学博士（医学）・学位論文・平成24年3月31日授与（甲第916号）・唐澤文寿Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal alpha 1,4-linked N-acetylglucosamine residues (alpha GlcNAc). Previously, we identified human alpha 1,4-N-acetylglucosaminyltransferase (alpha 4GnT), which is responsible for the O-glycan biosynthesis and characterized alpha GlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(-/-) mice to better understand its role in vivo. A4gnt(-/-) mice showed complete lack of alpha GlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(-/-) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced alpha GlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of alpha GlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, alpha GlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation.ArticleJOURNAL OF CLINICAL INVESTIGATION. 122(3):923-934 (2012)journal articl

Comparison of Methods to Release Mucin-Type O‑Glycans for Glycomic Analysis

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called “peeled” products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans

Immunostaining of the gastric corpus (A–E) and antral (F–J) mucosae with anti-mucin monoclonal antibodies

Gastric tissues were obtained from control rats (A, F), rats treated with 5-fluorouracil (5-FU) alone (B, G), rats treated with omeprazole (Ome+ 5-FU (C, H), rats treated with lansoprazole (Lan)+5-FU (D, I), and rats treated with lafutidine (Laf)+5-FU (E, J). Notice that surface epithelial mucus cells in the corpus show positive staining with RGM21, and those in the antrum show positive staining with RGM26. Original magnification ×25.<p><b>Copyright information:</b></p><p>Taken from "Effects of acid antisecretory drugs on mucus barrier of the rat against 5-fluorouracil-induced gastrointestinal mucositis"</p><p></p><p>Scandinavian Journal of Gastroenterology 2008;43(5):531-537.</p><p>Published online Jan 2008</p><p>PMCID:PMC2430178.</p><p></p

Essential role of gastric gland mucin in preventing gastric cancer in mice

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt(–/–) mice to better understand its role in vivo. A4gnt(–/–) mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt(–/–) mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation

Immunostaining of the rat jejunal (A–E) and ileal (F–J) mucosae with anti-mucin monoclonal antibody PGM34

Small-bowel tissues were obtained from control rats (A, F), rats treated with 5-fluorouracil (5-FU) alone (B, G), rats treated with omeprazole (Ome)+5-FU (C, H), rats treated with lansoprazole (Lan)+5-FU (D, I), and rats treated with lafutidine (Laf)+5-FU (E, J). Notice that goblet cells in the jejunum and ileum show positive staining with PGM34. Original magnification ×25.<p><b>Copyright information:</b></p><p>Taken from "Effects of acid antisecretory drugs on mucus barrier of the rat against 5-fluorouracil-induced gastrointestinal mucositis"</p><p></p><p>Scandinavian Journal of Gastroenterology 2008;43(5):531-537.</p><p>Published online Jan 2008</p><p>PMCID:PMC2430178.</p><p></p

Influence of acid antisecretory agents on the jejunal (A) and ileal (B) mucin accumulation in the 5-FU-induced small-bowel mucosal damage

Fr-1 hexose values corresponding to mucin content are expressed as micrograms of hexose per rat and represent means±SD. Abbreviations: 5-FU = 5-fluorouracil; Ome = omeprazole; Lan = lansoprazole; Laf = lafutidine. = 6–9 (each group); *<p><b>Copyright information:</b></p><p>Taken from "Effects of acid antisecretory drugs on mucus barrier of the rat against 5-fluorouracil-induced gastrointestinal mucositis"</p><p></p><p>Scandinavian Journal of Gastroenterology 2008;43(5):531-537.</p><p>Published online Jan 2008</p><p>PMCID:PMC2430178.</p><p></p